ABSTRACT Retroviral vectors are widely used in gene therapy. However, a recent gene therapy trial demonstrated that integration of a retroviral vector into host cell genes may lead to development of cancer. Therefore, control of integration site selection is critical to a successful outcome of gene therapy approaches, which use these vectors. This grant proposal is based on a new work, which implicates a host cell transcription factor and a histone modifier protein in integration site selection by retroviral vectors, and focuses on the development of new methods to target integration to predetermined chromosomal regions. Results supporting a role of these proteins in targeting were published very recently, and new preliminary data are presented in this proposal. These data suggest that transcription factor-based synthetic proteins can be used to control integration site selection. The histone modifier results are novel and not yet published. In Aim 1, genetic and molecular biology approaches are employed to determine the integration rate in and outside transcription units in the presence of high intracellular amounts of the synthetic proteins. It is proposed that a synthetic gene consisting of the heterochromatin protein 1 alpha (HP-1 chromatin-binding domain and the transcription factor 3'-terminus will ) be transiently expressed and the frequency of integration inside and outside genes will be determined using the recently completed human genome sequence and novel Alu-PCR- and ChIP-based methods. Similarly, another transcription factor-based gene will be constructed, where the HP-1 chromatin-binding domain will be replaced by a DNA-binding domain of another transcription factor. The above mentioned methods will be used to determine if this novel fusion gene targets integration to a specific DNA sequence. In the Aims 2A and 2B, it will be determined whether the histone modifier protein and its activity targets integration away from genes. Experiments, which are proposed in the last sub-Aim, 2C, aim to maximize the efficiency of the integration targeting to heterochromatin by simultaneous expression of transcription factor- based and histone modifier proteins. Results from these and related experiments will have important implications for gene therapy approaches using retroviral vectors, since they will lead to novel therapeutic approaches that will decrease the danger of cancer development due to integration in undesirable regions of the human genome. Finally, our results should also significantly enhance our understanding of the role of chromatin modifications in integration site selection by retroviral vectors. NARRATIVE Retroviruses are widely used as vehicles to accomplish the delivery of therapeutic genes into target cells, in a process called integration. In some cases, integration may happen in an undesirable region of a patient's DNA, and as a result the patient may develop cancer. In this grant application, we propose two independent approaches, which should target integration away from potentially dangerous regions of human DNA, and lead to integration in regions that are safe from the possibility of cancer development.